The present invention relates to a process for producing and correlating light microscope images and spectroscopic data resolved according to wavelength of a sample by means of confocal scanning light-microscopy and spectrometers.
It is known practice (Applied Physics 22 (1980), p.119) to produce light microscope images of transparent or semitransparent test objects using scanning light microscopy. If the beam path corresponds to the principle of the confocal light microscope (CLSM), optical sectional images are obtained, i.e. images of a narrow zone around the focal plane of the microscope lens. If the focal plane lies within the tested object, then owing to the confocal principle the intensities from the regions of the sample lying above and below are to a large extent eliminated. For some years now, appliances operating on said principle have been commercially available.
It is a further known practice to acquire data about the chemical structure of a test object by examining it using spectroscopic techniques, in particular by using light from the visible region of the spectrum or from regions close to the visible spectrum.
The combination of light-microscopy and spectroscopic techniques is also known from literature:
A concept already realized by most manufacturers of commercial confocal light microscopes is that of producing diffuse reflection images in the fluorescence contrast. For this purpose, the sample is illuminated by a monochromatic light source. There is inserted into the imaging beam path an optical filter which as completely as possible retains the light having the wavelength of the illuminating light source, so that only the fluorescent light having a longer wavelength reaches the photoelectric detector.
It is further known, from Microscopia Acta 79 (1977), 3, p.267-276, to add to the imaging beam path of a conventional light microscope a spectrograph which has been adjusted to a specific Raman line of a selected substance present in the test object. In said manner, a microscopic Raman dark field image of the object is produced. Said image reproduces the local distribution of the selected substance in the test object.
From Nature 347, (20.09.1990) p.301-303, it is known practice to use the concept of the confocal optical beam path in order to be able to record the complete Raman spectrum of a small pre-selectable measuring volume within the test object. For said purpose, the test object is illuminated by a laser by way of a stationary arrangement according to the concept of the confocal beam path, and the light passing through the aperture of the imaging beam path is analyzed in a Raman spectrometer. In said manner, the Raman spectrum of a selected measuring volume in the order of magnitude of 1 .mu.m**3 is obtained.
The drawback of all these processes known from literature which combine light-microscopy and spectroscopic techniques is that it is impossible to exploit the full potential of both techniques simultaneously:
The standard process of confocal fluorescence microscopy used up till now utilizes only the mean intensity of fluorescence transmitted by the measuring filter to build up the image. The fine details of the fluorescence spectrum are on the other hand not utilized. The process according to Microscopia Acta 79 (1977), 3, p.267-276, does not supply three-dimensional microscopic data, like confocal light microscopy, but only two-dimensional images and, moreover, of the data available in the Raman spectra it uses only those of a previously selected line when producing an image.
The process according to Nature 247, p.301-303, because of the absence of scanning microscope image production, likewise does not offer any three-dimensional microscopic data but does provide the entire Raman spectrum of the examined sample volume.